Mechanistic investigation of protein translocation on DNA

化学系的生命过程化学项目支持加州大学圣巴巴拉分校的Norbert Reich教授研究细胞蛋白质如何定位DNA上的特定位置。众所周知，DNA储存着遗传信息，这些信息在细胞内的其他生物分子中被转录和翻译，这强调了每个细胞的生命。DNA信息的“阅读和解释”需要与细胞中的其他分子，特别是蛋白质相互作用，并受其调节。通过蛋白质寻找DNA上的特定位置是具有挑战性的，因为DNA看起来非常均匀。这个建议的重点是蛋白质如何识别DNA上要结合的特定位置，然后它们如何在它们结合的DNA上易位。这项工作将对科学家确定存储在DNA中的信息如何被有效解释的能力产生更广泛的影响，这是所有细胞共同的过程。它正在进一步影响下一代科学家的教育，包括本科生和研究生，他们有能力认识和解决与人类健康有关的科学问题。此外，Reich教授和他的学生积极参与旨在向广大观众解释科学如何运作以及科学给社会带来什么好处的活动。作用于DNA的蛋白质负责DNA的绝大部分功能。这项提议的首要目标是提供对细菌DNA腺嘌呤甲基转移酶（Dam，修饰5'-GATC-3'）这种蛋白质的更深入了解。Dam及其相关酶进行多循环催化（过程催化）的能力对其生物学作用至关重要。然而，目前的过程催化模型主要是基于面临完全不同的生物学挑战的酶，并没有考虑到这些酶的特性。例如，DNA甲基转移酶有效地修饰具有高度可变位点间距离的多个识别位点，并且这种过程通过相互作用的蛋白质和靶位点两侧的DNA序列来调节。本研究的目的是探讨Dam过程催化的机制、调控及其在体内的重要性。将采用几种策略来解决广泛相关且未解决的问题，如Dam和其他蛋白质如何在超出传统模型预测的位点间距离上与DNA底物进行过程性作用，一些蛋白质（如Dam）如何依赖片段内转移来有效地进行长距离移动，以及单体Dam在不离开DNA的情况下修饰两条DNA链的机制是什么。

The Chemistry of Life Processes Program in the Division of Chemistry is supporting Professor Norbert Reich of University of California Santa Barbara to investigate how cellular proteins locate specific positions on DNA. It is well known that DNA stores genetic information that is transcribed and translated within cells in other biological molecules and that underscores the life of every cell. The "reading and interpretation" of the DNA information require and are modulated by interactions with other molecules in the cell, particularly proteins. The search by proteins of specific locations on DNA is challenging because DNA appears very uniform. This proposal focuses on how the proteins identify specific locations on DNA to bind to and then how they translocate on the DNA to which they are bonded. This work will have a broader impact on scientists' ability to determine how the information stored in DNA is productively interpreted, which is a process common to all cells. It is having further impact on the education of the next generation of scientists, both undergraduate and graduate students, capable of recognizing and addressing scientific issues relevant for human health. Furthermore, Professor Reich and his students actively engage in activities aimed at explaining to broad audiences how science works and what benefits science brings to society.Proteins that act on DNA are responsible for the vast majority of the functions of DNA. The overarching goal of this proposal is to provide a deeper understanding of one such protein, the bacterial DNA adenine methyltransferase (Dam, modifies 5'-GATC-3'). The ability of Dam and related enzymes to carry out multiple cycles of catalysis (processive catalysis) is critical to their biological roles. Yet, current models of processive catalysis are based largely on enzymes faced with entirely different biological challenges and do not account for the characteristics of these enzymes. For example, DNA methyltransferases efficiently modify multiple recognition sites with highly variable intersite distances, and this processivity is modulated by interacting proteins and DNA sequences that flank the target sites. The aims of the research are to investigate the mechanism, regulation, and in vivo importance of processive catalysis by Dam. Several strategies will be pursued to address broadly relevant and unanswered questions of how Dam and other proteins act processively with DNA substrates over intersite distances beyond the ones predicted by conventional models, how some proteins, such as Dam, rely on intra-segment transfer to efficiently move large distances, and what is the mechanism whereby the monomeric Dam modifies both strands of DNA without leaving the DNA.